CN110698386A - Preparation and application of pH near-infrared fluorescent probe - Google Patents

Abstract

The invention relates to the preparation and application of a pH near-infrared fluorescent probe. The structural formula of the fluorescent probe is:

The invention provides a preparation method for synthesizing the fluorescent probe using 1,1,2-trimethyl-1H-benzo[e]indole, cyclohexanone, iodoethane and the like as raw materials; the fluorescent probe is a A pH near-infrared fluorescent probe; firstly, the fluorescent probe exhibits high sensitivity in an acidic environment; secondly, the fluorescent probe exhibits high selectivity to pH and is not disturbed by other inorganic ions; and , the fluorescent probe responds very quickly to pH; in addition, the fluorescent probe is used to distinguish normal cells from cancer cells.

Description

Preparation and application of a pH near-infrared fluorescent probe

technical field

The invention belongs to the technical field of fluorescent probes, in particular to the preparation and application of a pH near-infrared fluorescent probe based on cyanine dyes.

Background technique

Intracellular pH plays a key role in many cellular events, including cell growth and apoptosis, ion transport and homeostasis, calcium regulation, endocytosis and cell adhesion, among others (Tang B, Yu F, Li P, et al. . Journal of the American Chemical Society, 2009, 131:3016-3023). Under normal physiological conditions, the concentration of extracellular hydrogen ions is kept within a very narrow range, and even small changes will cause many diseases (Lagadic-Gossmann D, Rissel M, Galisteo M, et al. British journal of pharmacology, 1999, 128:1673-1682.). Previous evidence suggests that the internal environment of tumors has been acidified (Schornack PA, Gillies RJ. Neoplasia, 2003, 5:135-145). As glucose metabolism increases, H ⁺ production and excretion in cancer typically also increases. Cellular pH (pH 6.5-6.9) is lower in malignant tumors under physiological conditions compared to normal tissues (pH 7.2-7.4) (Stubbs M, McSheehy PMJ, Griffiths JR, et al. Molecular medicine today, 2000, 6 : 15-19; Van Sluis R, Bhujwalla ZM, Raghunand N, et al. Magnetic Resonance in Medicine: An Official Journal of the International Society for Magnetic Resonance in Medicine, 1999, 41:743-750). Therefore, we can regard pH as an effective cancer biomarker and a breakthrough for early detection of cancer, which urgently needs to design effective methods to accurately detect it.

Fluorescence detection methods have received extensive attention due to their high sensitivity, simple operation, and application in biological imaging. In recent years, many fluorescent probes have been designed and developed to detect pH, such as coumarin-based probes (Dong B, Song X, Wang C, et al. Analytical chemistry, 2016, 88:4085-4091), Hemicyanine-based probes (Wu L, Wang Y, James T D, et al. Chemical communications, 2018, 54:5518-5521;) and pyrene-based probes (Cao L, Zhao Z, Zhang T, et al . Chemical Communications, 2015, 51: 17324-17327). However, these probes have shorter excitation and emission wavelengths (<550 nm), resulting in excessive autofluorescence and shallow penetration depth, which reduces the sensitivity of the probes and hinders their application in biological systems. In contrast, near-infrared (NIR) fluorescent probes have little photodamage and can penetrate deeply into tissues, thereby minimizing the interference of background fluorescence, which is more beneficial for biological imaging. Therefore, it is of great interest to design and synthesize near-infrared fluorescent probes with long-wavelength emission.

Cyanine dyes are near-infrared fluorescent dyes. At present, fluorescent probes based on cyanine dyes have been used to detect Hg ²⁺ , NO, H ₂ O ₂ , and ozone, etc. (Guo Z, Zhu W, Zhu M, et al.Chemistry-A European Journal , 2010, 16: 14424-14432; Sasaki E, Kojima H, Nishimatsu H, et al. Journal of the American Chemical Society, 2005, 127: 3684-3685; Yu F, Li P, Song P, et al. Chemical communications, 2012 , 48:4980-4982; Xu K, Sun S, Li J, et al. Chemical Communications, 2012, 48:684-686). However, very few probes based on cyanine dyes have been used to detect pH. Therefore, it is necessary to design and synthesize a near-infrared fluorescent probe based on cyanine dyes to detect pH and differentiate normal cells from cancer cells.

SUMMARY OF THE INVENTION

According to the proposed requirements, the present inventors have conducted in-depth research on this, and after a lot of creative work, provide a pH near-infrared fluorescent probe based on cyanine dyes.

The technical scheme of the present invention is, a kind of pH near-infrared fluorescent probe, and its structural formula is as follows:

A preparation method of pH near-infrared fluorescent probe. Proceed as follows:

In a 100 mL round-bottomed flask, dissolve 1 equivalent of cyanine CyCl and 2 to 3 equivalents of sodium acetate into 5 to 10 mL of N,N-dimethylformamide, under nitrogen protection, and stir for 10 to 14 h to stop the reaction. Then, the reaction mixture was cooled to room temperature, extracted with dichloromethane, and the organic layer was washed with saturated brine. The organic layer was dried with anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The crude product was subjected to column chromatography with CH ₂ Cl ₂ /CH ₃ CH ₂ OH eluent in a volume ratio of 100:1 to 20:1 to obtain a red color The solid product (yield 52%) was the fluorescent probe.

The beneficial effect of the invention is that a pH near-infrared fluorescent probe has good spectral response performance. First, the fluorescence spectral properties of the probe were studied. Under neutral conditions, the fluorescent probe had no near-infrared (780nm) fluorescence emission peak; under acidic conditions, a fluorescence emission peak appeared in the near-infrared region (780nm). And with the enhancement of acidic conditions, the near-infrared fluorescence intensity of the probe molecules increased continuously. Therefore, the probe can detect pH under acidic conditions. Secondly, the UV absorption spectrum of the probe was studied. Under neutral conditions, the probe had an absorption band at 560 nm; with the increase of acidic conditions, the absorption peak at 560 nm gradually decreased, and a new absorption peak appeared near 760 nm. Then, the selectivity of the probe was studied, and the probe and inorganic ions (K ⁺ , Ca ²⁺ , Na ⁺ , Mg ²⁺ , Fe ²⁺ , Fe ³⁺ , Cr ³⁺ , Hg ²⁺ , HCO ₃ ^- , F ^- , Br ^- , Ac ^- , SO ₄ ^2- , NO ₃ ^- .) fluorescence response at pH=5.0 and pH=7.4. It was found that only pH could change the fluorescence spectrum, and other inorganic ions had no obvious effect on the fluorescence spectrum of the probe. In addition, this fluorescent probe responds very rapidly to pH.

Application of a pH near-infrared fluorescent probe. When fluorescent probes were added to normal cells, almost no fluorescence was observed, indicating that the pH in normal cells was neutral. When fluorescent probes were added to cancer cells, strong fluorescence was observed, indicating that the pH in cancer cells was low. These results indicate that fluorescent probes can distinguish normal cells from cancer cells, which provides a reliable means for early detection of cancer.

Description of drawings

Figure 1 shows the synthetic route of the fluorescent probe.

Figure 2 shows the UV-Vis absorption spectra of fluorescent probes in different pH buffers.

The abscissa is the wavelength, and the ordinate is the absorbance. The concentration of fluorescent probe was 5 μM, the concentration of SDS was 5 mM, and the pH was: 5.0, 5.5, 5.9, 6.2, 6.5, 6.8, 7.4, respectively.

Figure 3 shows the fluorescence spectra of fluorescent probes in different pH buffers.

The abscissa is the wavelength, and the ordinate is the fluorescence intensity. The concentration of fluorescent probe was 5 μM, the concentration of SDS was 5 mM, and the pH was: 5.0, 5.5, 5.9, 6.2, 6.5, 6.8, 7.4, respectively. The fluorescence excitation wavelength was 720 nm.

Figure 4 is a graph of the fluorescence linear response of fluorescent probes in different pH buffers.

The concentration of fluorescent probes was 5 μM, and the concentration of SDS was 5 mM. The fluorescence excitation wavelength was 720 nm.

Figure 5 is a graph of the selectivity of fluorescent probes.

The concentrations of fluorescent probes are all 5μM, the concentration of SDS is 5mM, and the concentrations of other analytes are all 100μM, respectively: 1.Blank, 2.K ⁺ , 3.Ca ²⁺ , 4.Na ⁺ , 5.Mg ²⁺ ,6.Fe ²⁺ ,7.Fe ³⁺ ,8.Cr ³⁺ ,9.Hg ²⁺ ,10.HCO ₃ ^- ,11.F ^- ,12.Br ^- ,13.Ac ^- ,14.SO ₄ ^2- ,15.NO ₃ ^- .

FIG. 6 is a graph showing the relationship between the fluorescence intensity of the fluorescent probe and the time change in different pH buffer solutions.

Figure 7 is a toxicity test for human colon cancer cells. The abscissa is the concentration of fluorescent probe, and the ordinate is the cell viability.

Figure 8 is a human colonic mucosal cytotoxicity assay. The abscissa is the concentration of fluorescent probe, and the ordinate is the cell viability.

Figure 9. Cell imaging of fluorescent probes in normal cells and cancer cells.

Detailed ways

The present invention is described in detail below with reference to the accompanying drawings and specific embodiments, but is not limited thereto.

Example 1:

Synthesis of Fluorescent Probes

The synthetic route is shown in Figure 1. In a 100 mL round-bottomed flask, dissolve 1 equiv of cyanine CyCl and 2 equiv of sodium acetate into 8 mL of N,N-dimethylformamide, under nitrogen protection, stir for 12 h, stop the reaction, and then cool the reaction mixture After reaching room temperature and extracting with dichloromethane, the organic layer was washed with saturated brine. The organic layer was dried with anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The crude product was subjected to column chromatography with CH ₂ Cl ₂ /CH ₃ CH ₂ OH eluent in a volume ratio of 100:1 to 20:1 to obtain a red color The solid product (yield 52%) was the fluorescent probe. ¹ H NMR (400 MHz, CDCl ₃ , ppm): δ 8.33 (d, J=12.8 Hz, 2H), 8.04 (d, J=8.8 Hz, 2H), 7.80 (d, J=8.4 Hz, 2H), 7.76(d,J＝8.4Hz,2H),7.47(t,J＝7.6Hz,2H),7.25-7.28(m,2H),7.07(d,J＝8.4Hz,2H),5.51(d,J =13.2Hz, 2H), 3.82-3.85(m, 4H), 2.63-2.66(m, 4H), 2.00(s, 12H), 1.93-1.87(m, 2H), 1.33(t, J=6.8Hz, 6H). ¹³ C NMR (100MHz, CDCl ₃ , ppm): δ 186.28, 163.9, 141.1, 132.9, 130.0, 130.0, 129.5, 129.1, 126.8, 126.3, 122.5, 121.9, 109.0, 91.9, 48.6, 37.2, 29.7, 28 , 25.9, 22.8, 11.5. MS(TOF): 591.4.

Example 2:

Fluorescent probe, SDS solution and different pH solution preparation

Preparation of probe solution: Weigh a certain amount of probe and dissolve it in dimethyl sulfoxide to prepare a 4×10 ^-4 M probe solution. Preparation of SDS solution: Weigh a certain amount of sodium dodecyl sulfate and dissolve it in pure water to prepare a 4×10 ^-1 M SDS solution. Preparation of different pH buffer solutions: Weigh a certain amount of NaCl, KCl, Na ₂ HPO ₄ and NaH ₂ PO ₄ , dissolve them in pure water, and measure by pH meter to prepare different pH buffer solutions.

Example 3:

Determination of UV-Vis Absorption Spectra of Fluorescent Probes in Different pH Buffers

Figure 2 shows the UV-Vis absorption spectra of fluorescent probes in different pH buffers. The concentration of fluorescent probes is 5 μM, the concentration of SDS solution is 5 mM, and the pH values are 5.0, 5.5, 5.9, 6.2, 6.5, 6.8, 7.4. The instrument used for UV-Vis absorption spectroscopy was an Agilent Cary60 UV-Vis spectrophotometer. It can be seen from Figure 2 that with the decrease of pH, the absorption peak of the probe at 560 nm gradually decreases, and the absorption peak at 760 nm gradually increases.

Example 4:

Determination of Fluorescence Spectra of Fluorescent Probes in Different pH Buffers

Figure 3 shows the fluorescence spectra of the fluorescent probes in different pH buffers. The concentration of the fluorescent probe is 5 μM, the concentration of the SDS solution is 5 mM, and the pH values are 5.0, 5.5, 5.9, 6.2, 6.5, 6.8, and 7.4. The excitation wavelength was fixed at 720 nm, and the emission wavelength ranged from 750 to 840 nm. The slit width is 5.0 nm/5.0 nm, and the fluorescence measuring instrument used is Hitachi F4600 fluorescence spectrophotometer. It can be seen from Figure 3 that with the decrease of pH, a fluorescence emission peak gradually appeared in the near-infrared region (780 nm), and its fluorescence intensity increased continuously. This is because a pull-push π-conjugated system is formed in this cyanine dye. Therefore, the probe can detect pH under acidic conditions. Figure 4 is a graph of the linear response of the probe under different pH buffers. It can be found that the fluorescence intensity has a linear relationship with different pH.

Example 5:

Selectivity of fluorescent probes for pH determination

Figure 5 is a graph of the selectivity of fluorescent probes for pH determination. Investigate the addition of SDS (5mM) and its 100μM inorganic ions (K ⁺ , Ca ²⁺ , Na ⁺ , Mg ²⁺ , Fe ²⁺ , Fe ³⁺ , Cr ³⁺ , Hg ² to 5μM fluorescent probe solution) ⁺ , HCO ₃ ^- , F ^- , Br ^- , Ac ^- , SO ₄ ^2- , NO ₃ ^- .) fluorescence response at pH=5.0 and pH=7.4. It can be seen from Figure 5 that only pH can cause the change of the fluorescence spectrum, and other inorganic ions have no obvious effect on the fluorescence spectrum of the probe. These results indicate that the fluorescent probes have good selectivity for pH.

Example 6:

Determination of Response Time of Fluorescent Probe and pH

We studied the response time of fluorescent probes to pH, and the results are shown in Figure 6. It can be seen from the figure that the response time of the probe to pH is very short, which can meet the requirements of real-time monitoring in real samples. It can also be seen from Fig. 6 that after the fluorescence intensity reaches the maximum value, the fluorescence intensity does not change in the following time, which indicates that the fluorescent probe has good photostability.

Example 7:

Application of Fluorescent Probes in Living Cells

First, we did a cytotoxicity assay, as shown in Figures 7 and 8. When 0-50 μM probe was added, the cell survival rates of human colon cancer cells and human colon mucosal cells were both above 90%. This can indicate that the fluorescent probe is less toxic. Then, we studied the application of fluorescent probes in living cells, and selected human colon cancer cells HCT116 and human normal colorectal mucosal cells FHC for confocal microscopy imaging. The results are shown in Figure 9. Adding fluorescent probes to the two types of cells respectively, it can be found that almost no fluorescence can be observed in normal cells, while strong fluorescence can be observed in cancer cells, which indicates that the pH in cancer cells is higher than that in normal cells. low pH. These results indicate that fluorescent probes can distinguish normal cells from cancer cells, which provides a reliable means for early detection of cancer.

Claims (3)

1. A pH near-infrared fluorescent probe having the following structure:

2. the preparation method of a kind of pH near-infrared fluorescent probe according to claim 1, is characterized in that, reaction step is as follows: In a 100 mL round-bottomed flask, dissolve 1 equivalent of cyanine CyCl and 2 to 3 equivalents of sodium acetate into 5 to 10 mL of N,N-dimethylformamide, under nitrogen protection, and stir for 10 to 14 hours to stop the reaction , then, the reaction mixture was cooled to room temperature, extracted with dichloromethane, the organic layer was washed with saturated brine, dried with anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The volume ratio of the crude product was 100:1 Column chromatography was performed with CH ₂ Cl ₂ /CH ₃ CH ₂ OH eluent of ~20:1 to obtain a red solid product, which is the fluorescent probe. 3 . The application of a pH near-infrared fluorescent probe according to claim 1 , wherein the fluorescent probe is used to distinguish normal cells from cancer cells. 4 .

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